Epigenetic investigation into circulating microRNA 197-3p in sera from patients affected by malignant pleural mesothelioma and workers ex-exposed to asbestos

The epigenetic role of microRNAs is established at both physiological and pathological levels. Dysregulated miRNAs and their targets appear to be a promising approach for innovative anticancer therapies. In our previous study, circulating miR-197-3p tested dysregulated in workers ex-exposed to asbestos (WEA). Herein, an epigenetic investigation on this circulating miRNA was carried out in sera from malignant pleural mesothelioma (MPM) patients. MiR-197-3p was quantified in MPM (n = 75) sera and comparatively analyzed to WEA (n = 75) and healthy subject (n = 75) sera, using ddPCR and RT-qPCR techniques. Clinicopathological characteristics, occupational, non-occupational information and overall survival (OS) were evaluated in correlation studies. MiR-197-3p levels, analyzed by ddPCR, were significantly higher in MPM than in WEA cohort, with a mean copies/µl of 981.7 and 525.01, respectively. Consistently, RT-qPCR showed higher miR-197-3p levels in sera from MPM with a mean copies/µl of 603.7, compared to WEA with 336.1 copies/µl. OS data were significantly associated with histologic subtype and pleurectomy. Circulating miR-197-3p is proposed as a new potential biomarker for an early diagnosis of the MPM onset. Indeed, miR-197-3p epigenetic investigations along with chest X-ray, computed tomography scan and spirometry could provide relevant information useful to reach an early and effective diagnosis for MPM.

Receiver Operating Characteristic (ROC) analysis was then used to quantify the accuracy of RT-qPCR results in discriminating data in significant comparisons. The ROC AUC for the comparative analysis of miR-197-3p levels in MPM vs. WEA was 0.62 (P = 0.014*; 95% confidence interval 0.525 to 0.708) (Fig. 2B), indicating that miR-197-3p levels significantly discriminate MPM from WEA, while the comparison between MPM and HS was not significant (AUC = 0.56, P = ns, 95% confidence interval 0.472 to 0.657) (Fig. 2C).
As shown in Supplementary Figure S1, linear regression analysis indicated a significant correlation between ddPCR and RT-qPCR values within both study groups. The R-square was 0.879 for miR-197-3p (P < 0.0001****).
MiR-197-3p expression, assessed by both PCR techniques, did not show significant dysregulation among sera from the three different MPM subtypes, such as epithelioid, sarcomatoid and biphasic, (Fig. 3).   Fig. 4A-B, results indicate that OS was not influenced by miR-197-3p expression (P > 0.05). Then, the impact of histological subtype on OS was evaluated. As shown in Fig. 4C, histological subtype (log-rank P = 0.0056**) significantly influenced OS: specifically, OS was longest in patients with epithelioid MPM (40.7%), intermediate in biphasic cases (28.6%), while the shortest OS was revealed in the sarcomatoid MPM subtype (15%).
Subsequently, Kaplan-Meier methodology was employed to determine the impact of pleurectomy on OS in all patients. OS was significantly influenced by pleurectomy (log-rank P = 0.0394*). OS was longest in patients who underwent pleurectomy (62.5%) compared to unoperated patients (25.4%) (Fig. 4D). Finally, the same methodology reported no correlation between OS and asbestos exposure (P > 0.05) or smoking status (P > 0.05) (Supplementary Figure S2).
Multiple linear regression analysis of miR-197-3p expression, related to independent variables, i.e. MPM histological subtypes, asbestos exposure, tobacco smoking status and surgical interventions (Table 1), gave no

Discussion
Many investigations indicate that miRNAs can be considered promising biomarkers for MPM, as well as for other cancers. Indeed, they are detectable in cells, tissues and body fluids of subjects/patients exposed to toxic agents, affected by inflammatory disorders and infectious diseases 27 . Identifying such sensitive and specific MPM biomarkers, alongside markers of asbestos exposure in workers who had previously been exposed to asbestos fibers (WEA), appears of paramount importance for this fatal cancer 9,35 . To this aim, several studies investigated the diagnostic value of tissue, pleural effusion, and circulating micro-RNA for MPM 27 . By using microRNA array or public databases, some microRNAs with potential diagnostic significance have been identified. Specifically, mir-126 36,37 , miR-103a-3p 38,39 and mir-2053 40 represent the most promising candidates for a MPM diagnosis. However, their diagnostic accuracy, assessed with the ROC curve, is still moderate.
In this study, circulating miR-197-3p was investigated as a potential MPM biomarker, in a significantly largesized serum sample made up of two cohorts, MPM and WEA. MiR-197-3p, whose gene maps in chromosome 1p13.3, has been reported as being involved in key cellular processes, such as cell proliferation, apoptosis, differentiation, metastasis and drug resistance 41 . Significant miR-197-3p dysregulation has been detected in sera and cells in different human tumors [42][43][44] . In addition, miR-197-3p has been demonstrated to function as a pro-or anti-oncogenic gene regulator depending on its target gene and target cell lineage 45,46 .
Herein, miR-197-3p was found to be significantly up-regulated in sera from MPM vs. WEA. Interestingly, the two PCR techniques used in this study, ddPCR and RT-qPCR, reported almost overlapping results. Nevertheless, our data showed, as expected, higher sensitivity and specificity for ddPCR than RT-qPCR. These results are consistent with other studies outlining ddPCR as an innovative technique, with lower sensitivity to PCR inhibitors, greater analytical power and reliability in quantifying rare targets from very limited clinical samples 59 .
It is worth noting that ddPCR is an improvement on conventional PCR technique, as it allows for more sensitive, accurate quantification of target nucleic acids 60 . DdPCR overcomes problems of potential discrepancies in PCR analyses: (i) ddPCR performs absolute quantification thanks to the principles of sample partitioning and Poisson statistics, thus overcoming normalization and calibrator issues; (ii) it is relatively insensitive to potential PCR inhibitors; (iii) it is characterized by more precision and sensitivity in detecting low target copies; (iv) it directly provides the result of the analysis expressed as the number of copies of target per microliter of reaction 34 .
The up-regulation of circulating miR-197-3p observed in MPM suggests that miR-197-3p may act as an activated oncogene. On the other hand, a recent study showed the down-regulation of miR-197-3p in hepatocellular carcinoma (HCC) tissues associated with tumor aggressiveness, indicating that this microRNA may be a predictor of prognosis in patients with HCC 30 . Moreover, it has been reported that miR-197-3p plays an In the present work, despite the similarity of miR-197-3p expression levels in MPM and HS sera, this circulating miR seems to be well suited as MPM marker. Indeed, it is unlikely that a putative increase of the miR-197-3p level over time, which can be revealed in an asbestos ex-exposed subject, indicates a "return to normal." As reiterated in the literature, asbestos fibers are never eliminated from the body, once inhaled 61 . An increase of miR-197-3p level in an asbestos-exposed subject could indicate the MPM onset at its early stage. Therefore, miR-197-3p increased level may allow the subject to be monitored more closely than other WEA, where this miR remains stable, to identify the occurrence of the MPM onset. Indeed, ROC curve analysis demonstrates AUCs (0.62/0.65) at the limit of acceptability for a marker, compared with what is reported in the literature (AUC > 0.70-0.80).
In a previous study, we reported that the expression of circulating miR-197-3p was significantly downregulated in sera from workers who had previously been exposed to asbestos (WEA) compared to HS 33 and its expression level was inversely correlated with cumulative exposure to asbestos, indicating that miR-197-3p can be considered a new biomarker for exposure to this tumorigenic mineral. Interestingly, comparing the data obtained from MPM reported in this study with those from WEA described in the aforementioned work, the expression of circulating miR-197-3p was higher in the MPM than in the WEA cohort, analyzed by both ddPCR and RT-qPCR, with statistical significance in ddPCR (Tukey's test, P = 0.0036**). The discrepancy between the two techniques is due to the RT-qPCR, which is less accurate than ddPCR.
It has been established that each microRNA, in addition to regulating a variable number of targets, is also regulated by several non-coding RNAs in a larger network of RNA-RNA interactions 62 . Indeed, miR-197-3p has been shown to have many upstream non-coding RNAs that regulate its expression levels. Some non-coding RNAs act by decreasing miR-197-3p levels through, for example, the sponging mechanism 63 . Others affect miR-197-3p expression by increasing its levels 51,64 . In both cases, variuos studies have observed the involvement of miR-197-3p regulations with drug resistance mechanisms 52,64 .
Therefore, it is interesting for the interpretation of our data to consider this intriguing aspect of miR-197-3p. Overall, MPM is difficult to diagnose as well as to treat. In recent years, strategies for the treatment of MPM have been evolving from chemotherapeutic agents, such as pemetrexed and cisplatin, to monoclonal antibodies or immune checkpoint inhibitors (ICI) 65 .
The significant increase in miR-197-3p levels found in the MPM cohort patients compared to WEA is at present under investigation, including studies related to some mechanism linked to drug resistance. Functional analyses may elucidate the role of miR-197-3p in the MPM onset.
In this study, we also confirm the importance of MPM histological subtypes for patient survival 66 , as the strongest and most consistent predictor of MPM survival. Similarly, we corroborate a significant correlation between patient survival and pleurectomy, as reported in other works 67 . However, our data indicate that miR-197-3p is not correlated with patient survival.
Overall, circulating miR-197-3p was found to be expressed at higher levels in MPM vs. WEA sera. Our results provide a new perspective on epigenetic alterations in the MPM, highlighting a possible role for miR-197-3p as a biomarker of this tumor, understood as a deviation from the WEA status. The interpretation of epigenetic alterations in MPM through miRNAs may contribute to the development of new diagnostic and therapeutic strategies. Further investigations are needed to elucidate its role in patients with MPM, including validation/ identification of its target genes and its involvement in asbestos-induced chronic inflammation. The challenge we face, is to understand the role of miR-197-3p and molecules interacting with it in the MPM onset.
It is worth noting that there is still a worrying lack of reliable diagnostic markers for an early MPM onset, prevention and screening, as well as validated targets for developing new therapeutic treatments. MiR-197-3p may represent a new marker to be analyzed both in WEA and MPM sera. Liquid biopsy can be obtained with low invasive methods and this miRNA appears to be a quantifiable, specific, sensitive biomarker.
In conclusion, circulating miR-197-3p could be added as minimally invasive screening test to follow-up WEA, that are individuals at high-risk to develop MPM.

Materials and methods
Sera. Serum samples (N = 225) were from malignant pleural mesothelioma affected patients (MPM, n = 75), workers ex-exposed to asbestos (WEA, n = 75) and healthy subjects (HS, N = 75). MPM sera were obtained from the Mesothelioma BioBank, Pathology Unit, City Hospital of Alessandria, Italy, whereas sera from WEA were supplied by the Occupational Medicine Unit, University of Ferrara, Ferrara, Italy, as previously described 33 . HS sera were collected at the Clinical Laboratory Analysis of the University Hospital of Ferrara, Italy. HS sera were taken from discarded laboratory samples before incineration, after routine analyses. HS were subjects in good health at the time of blood sampling, as reported in the hospital records 33 .
Sera were anonymously collected, aliquoted and stored at − 80 °C until the time of analysis. All peripheral blood samples (HS, MPM and WEA) were collected employing the same method, i.e. in tubes with a serum gel separator. Samples were allowed to clot at room temperature for 30 min, then, they were centrifuged at 1,500 g for 10 min at room temperature, within 2-4 h of blood draw to ensure miRNA stability and a good extraction output 68 . After centrifugation, the supernatant was aliquoted into 1.5-mL sterile RNase-free tubes and immediately frozen at − 80 °C until the day of miRNA extraction. For comparative analyses, sera of MPM, WEA and HS were coded with indications of age, gender and pathology, if any. The three cohorts were made up of subjects/ patients with similar ages (MPM 69.3 ± 0.85; WEA 66.3 ± 0.78 and HS 69.8 ± 0.92, years ± SD). All study participants signed their written informed consent at the time of the hospital admission. The study was approved by the County Ethical Committee, Ferrara, Italy (ID numbers 151078, 160,986).

Scientific Reports
| (2023) 13:6501 | https://doi.org/10.1038/s41598-023-33116-z www.nature.com/scientificreports/ Clinicopathological characteristics, occupational and non-occupational information, were collected from WEA 33 and MPM cohorts (Table 1 and 2). Specifically, for the WEA cohort, data related to asbestos exposure and tobacco smocking status were from our previous study 33 . For the MPM cohort, in addition to asbestos exposure and tobacco smoking status, collected information concern MPM histological subtypes, surgical interventions, pharmacological therapies and concurrent diseases, not related to asbestos.
Ethics approval and consent to participate. The study was conducted according to the guidelines of the Declaration of Helsinki (as revised in 2013). The Ethics Committee of Ferrara approved the study (ID numbers 151078, 160,986) and all study participants signed their written informed consent.
RNA extraction and miRNA reverse-transcription. Total RNA, including miRNAs, was extracted from 200 μl of serum from each patient/subject involved in the study. The miRNeasy Mini Kit (QIAGEN, RRID:SCR_008539, Milan, Italy) was used following the manufacturer's instructions, with the following minor modifications. The final amount of miRNA, extracted from serum, may be influenced either miRNA extraction efficiency or RT-qPCR robustness. These factors can be verified by synthetic non-human miRNAs added to the serum sample, employed as controls, before the RNA isolation. In order to adjust these parameters, synthetic cel-miR-39 miRNA (2.5 μl of 5 nM) (QIAGEN, RRID:SCR_008539, Milan, Italy) was added to the serum sample (200 ul), soon after adding the Qiazol Reagent (1 ml) to be used as an exogenous extraction control. RNA was eluted in 30 µl RNAse-free water.
Stock solutions (100 μM) of synthetic oligonucleotides, in RNase-free/DNase-free water, were prepared following the manufacturer's instructions. In order to construct the standard curve of synthetic oligonucleotides miR-197-3p, serial dilutions of the stock solution were performed at 1:10 from 10 13 copies/μl to 10 0 copies/μl. The obtained miR-197-3p standard curve was used to quantify miRNAs absolutely by RT-qPCR 70 and one point of the curve was used as a positive control in ddPCR.

Droplet digital PCR (ddPCR).
To allow for more specific, analytical detection of miR-197-3p copies/μl and in order to validate RT-qPCR results, the QX200ddPCR System (Bio-Rad Laboratories, RRID:SCR_008426, Hercules, CA USA) was used for ddPCR analyses as previously reported 71,72 .
Briefly, ddPCR was performed in a total volume of 22 μl by adding 11 μl of 2X EvaGreen supermix (Bio-Rad Laboratories, RRID:SCR_008426, Hercules, CA USA), 10 μl of diluted cDNA template (1:60), and 0.5 μl of the specific miRCURY LNA miRNA PCR Assay. After droplet generation inside the Automated Droplet Generator (Bio-Rad Laboratories, RRID:SCR_008426, Hercules, CA USA), the 96 well-plate was heat-sealed with foil and placed in the SimpliAmp Thermal Cycler (Thermo Fisher Scientific, RRID:SCR_008452, Waltham, MA USA). PCR conditions were: 95 °C for 5 min, then 40 cycles at 95 °C for 30 s and 56 °C for 1 min (ramping rate of 2 °C/ sec), and three final steps at 4 °C for 5 min, 90 °C for 5 min and a 4 °C indefinite hold to enhance dye stabilization. Each PCR experiment included a mock sample containing H 2 O as a negative control and the synthetic LNA oligonucleotide used as a positive control. Finally, the QX200 Droplet Reader (Bio-Rad Laboratories, RRID:SCR_008426, Hercules, CA USA), and Quanta Soft software (Bio-Rad Laboratories, RRID:SCR_008426, Hercules, CA USA), were used for data analyses.
The thermal cycler conditions were as follows: denaturation at 95 °C 2 min, followed by 40 cycles at 95 °C 10 s and 56 °C 1 min. A final melting curve step, from 56 °C to 95 °C (ramping rate of 0.5 °C/sec), was carried out to verify the amplified product. Standard curves were run in parallel with samples for absolute quantification of miR-197-3p.
Statistical analysis. Statistical analyses were performed using Prism 8.0 statistical software (GraphPad Prism, RRID:SCR_002798) version #8, La Jolla, CA, USA). Data distribution was verified using the D' Agostino Pearson test or the Shapiro-Wilk test, depending on the number of the cohort examined. As values did not follow a Gaussian distribution, we performed a log-transformation of data in order to carry out statistical analyses. Untransformed data have been shown for ease of interpretation. One-way analysis of variance (ANOVA) and Tukey's multiple comparisons tests were used to assess differences in the mean values of copies/µl of miR-197-3p in all study cohorts. The correlation between RT-qPCR and ddPCR data was tested using the linear regression model. ROC curves were used to evaluate the reliability of circulating miR-197-3p as a malignant pleural mesothelioma biomarker. All parameters, collected for the MPM cohort, were correlated with patients' overall survival (OS) at 18 months using the Kaplan-Meier model to assess the relationship between miR-197-3p and OS. High and low levels of miR-197-3p have been defined using the mean value of miR-197-3p expression to calculate the cut-off point. This is one of the multiple methods available to determine the cutoff point in overall survival analysis 73 . The Log-Rank Test was used to evaluate group differences. P values less than 0.05 were considered statistically significant for all analyses. Therefore, Spearman correlation coefficient was used for correlation analysis between miR-197-3p expression and independent variables, i.e. clinicopathological characteristics.

Data availability
The data presented in this study are available on request from the corresponding author.